association of trim22 with the type 1 interferon response

JOURNAL OF VIROLOGY, Jan. 2011, p. 208–216 Vol. 85, No. 10022-538X/11/$12.00 doi:10.1128/JVI.01810-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Association of TRIM22 with the Type 1 Interferon Responseand Viral Control during Primary HIV-1 Infection�

Ravesh Singh,1 Gaurav Gaiha,2 Lise Werner,3 Kevin McKim,2 Koleka Mlisana,3 Jeremy Luban,4Bruce D. Walker,1,2,5 Salim S. Abdool Karim,3 Abraham L. Brass,2 Thumbi Ndung’u,1,2,3*

and the CAPRISA Acute Infection Study TeamHIV Pathogenesis Programme, Doris Duke Medical Research Institute, Nelson R. Mandela School of Medicine, University of

KwaZulu-Natal, Durban, South Africa1; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute ofTechnology and Harvard University, Boston, Massachusetts2; Centre for the AIDS Programme of

Research in South Africa (CAPRISA), University of KwaZulu-Natal, Durban, South Africa3;Department of Microbiology, University of Geneva, Geneva, Switzerland4; and

Howard Hughes Medical Institute, Chevy Chase, Maryland5

Received 26 August 2010/Accepted 14 October 2010

Type 1 interferons (IFNs) induce the expression of the tripartite interaction motif (TRIM) family of E3ligases, but the contribution of these antiviral factors to HIV pathogenesis is not completely understood. Wehypothesized that the increased expression of select type 1 IFN and TRIM isoforms is associated with asignificantly lower likelihood of HIV-1 acquisition and viral control during primary HIV-1 infection. Wemeasured IFN-�, IFN-�, myxovirus resistance protein A (MxA), human TRIM5� (huTRIM5�), and TRIM22mRNA levels in peripheral blood mononuclear cells (PBMCs) of high-risk, HIV-1-uninfected participants andHIV-1-positive study participants. Samples were available for 32 uninfected subjects and 28 infected persons,all within 1 year of infection. HIV-1-positive participants had higher levels of IFN-� (P � 0.0005), MxA (P �0.007), and TRIM22 (P � 0.01) and lower levels of huTRIM5� (P < 0.001) than did HIV-1-negative partici-pants. TRIM22 but not huTRIM5� correlated positively with type 1 IFN (IFN-�, IFN-�, and MxA) (all P <0.0001). In a multivariate model, increased MxA expression showed a significant positive association with viralload (P � 0.0418). Furthermore, TRIM22 but not huTRIM5�, IFN-�, IFN-�, or MxA showed a negativecorrelation with plasma viral load (P � 0.0307) and a positive correlation with CD4� T-cell counts (P �0.0281). In vitro studies revealed that HIV infection induced TRIM22 expression in PBMCs obtained fromHIV-negative donors. Stable TRIM22 knockdown resulted in increased HIV-1 particle release and replicationin Jurkat reporter cells. Collectively, these data suggest concordance between type 1 IFN and TRIM22 but nothuTRIM5� expression in PBMCs and that TRIM22 likely acts as an antiviral effector in vivo.

Tripartite interaction motif (TRIM) E3 ligases represent arecently described family of proteins with potent antiviral ac-tivity (39). There are approximately 70 TRIM family members,and they are characterized by the presence of a tripartite motif,which consists of a RING domain, one or two B-box motifs,and a coiled-coil region (21, 29, 39). The presence of the RINGdomain suggests that these proteins function as E3 ubiquitinligases and mediate ubiquitylation events (6). The E3 ubiquitinligase activity of the RING domain is important for the anti-retroviral function of many TRIM proteins (29, 38).

The prototype member of this family, TRIM5�, is respon-sible for the complete block of HIV-1 replication in Old Worldmonkey cells (33, 36). This effect is mediated through theinteraction of rhesus monkey TRIM5� (TRIM5�rh) with theHIV-1 capsid (36). Further studies suggested that in additionto the effects of TRIM5�rh on HIV via binding to capsid, othermechanisms of viral inhibition are possible (27, 31). TRIM5�is responsible for the species-specific postentry restriction ofretroviruses, such as N-tropic murine leukemia virus (N-

MuLV) and HIV-1, in primate cells (36, 45). Other TRIM E3ligases with antiviral activity have been described (45). TRIMfamily proteins affect specific steps in the HIV life cycle (13).TRIM proteins appear to mediate their antiviral activities viadiverse mechanisms: interference with the uncoating of theviral preintegration complex was noted for TRIM5� (24), andan inhibition of viral budding has been described for TRIM22(26).

Although the antiretroviral activity of TRIM E3 ligases isestablished, the contribution of this family of proteins to pro-tection against HIV-1 infection or to the control of diseaseprogression is largely unknown. Many in vitro studies havesuggested that human TRIM5� (huTRIM5�) has little effecton HIV replication. However, some huTRIM5� genetic vari-ants have been associated with reduced susceptibility to HIVinfection (14, 35), suggesting that huTRIM5� may have a pro-tective role in infection. Modest effects of huTRIM5� geneticpolymorphisms on the rate of disease progression have alsobeen reported (9, 41), and it was suggested previously thathuman TRIM5� may select for HIV-1 escape mutants after aprolonged duration of infection (17).

In a prospective cohort study of HIV-1-negative individualsat high risk for HIV-1 infection, we have recently shown thatelevated levels of expression of huTRIM5� are associated withdecreased susceptibility to HIV-1 infection (34). Furthermore,

* Corresponding author. Mailing address: Doris Duke Medical Re-search Institute, University of KwaZulu-Natal, Private Bag X7, Con-gella, Durban 4001, South Africa. Phone: 2731 260 4727. Fax: 2731 2604623. E-mail: [email protected].

� Published ahead of print on 27 October 2010.

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we found that huTRIM5� mRNA levels were neither activelydownregulated nor upregulated among individuals in this co-hort who eventually became HIV-1 infected. The latter findingwas surprising, because previous studies demonstrated thattype 1 interferons (IFNs) are dysregulated during HIV-1 in-fection (10, 15), and TRIM5� is a well-known type 1 IFN-inducible gene (1, 31). Therefore, we would have expected toobserve trends for huTRIM5� similar to those reported fortype 1 IFN. In addition to TRIM5�, several TRIM proteinswith antiviral activity were recently described and were shownto be IFN inducible (2, 39). However, there are also notableexceptions to this IFN inducibility rule (4, 28). IFNs are them-selves the main mediators of innate immunity to viral infection,and they play a significant role by upregulating the expressionof many antiviral effectors within the cell (5, 30). Our under-standing of the type 1 IFN regulation of TRIMs is complicatedby the fact that IFN-� has 13 functional isoforms. It is unclearwhether all these isoforms have the same effect on IFN-stim-ulated genes (23).

Here we investigated the expression of the type 1 IFNsIFN-� (IFN-�2b isoform), IFN-�, a surrogate interferon-in-ducible gene (myxovirus resistance protein A [MxA]), andTRIM22 in a longitudinal cohort of black African females athigh risk for HIV-1 infection, for which we have previouslydemonstrated that enhanced TRIM5� mRNA expression isassociated with reduced susceptibility to HIV infection (34).TRIM22 was selected for this analysis because in addition toTRIM5�, it is one of the relatively well-characterized TRIME3 ligases, has been shown to be type 1 IFN inducible in vitro,and appears to possess anti-HIV-1 activity (2, 4, 25). Specifi-cally, we tested the hypotheses that the increased expression oftype 1 IFNs and TRIM22 is associated with a significantlylower likelihood of HIV-1 acquisition and lower viral loads orhigher CD4� T-cell counts during primary HIV-1 infection.We tested whether there are differences in mRNA levels of selecttype 1 IFNs, huTRIM5�, and TRIM22 in peripheral blood mono-nuclear cells (PBMCs) from HIV-negative versus HIV-positiveindividuals. We used multivariate analysis models in order tobetter understand the kinetics and antiviral implications of theexpression of these genes in vivo. Finally, we performed in vitroexperiments to better understand the relationship between type 1IFNs, TRIM22 expression, and antiviral activity.

MATERIALS AND METHODS

Subjects. Study subjects were part of the CAPRISA 002 Acute InfectionStudy, which is an observational natural history study of HIV-1 subtype Cinfection established in Durban, South Africa, in 2004 (34, 40). The cohortconsisted of 245 high-risk seronegative women who were monitored to identifyacute or recent infections. Participants were enrolled into the acute-infectionphase if they were antibody positive within 5 months of a previous antibody-negative test or if they had evidence of viral replication without HIV-1 antibod-ies, as assessed by rapid tests and PCR testing. Women from other seroincidentcohorts in Durban were enrolled into CAPRISA 002 if they met the above-described criteria. Time of infection was defined as the midpoint between the lastHIV antibody-negative test and the first HIV antibody-positive test or 14 daysprior to the first positive HIV RNA PCR assay for those identified as beingantibody negative but HIV RNA PCR positive. The study was approved by theBiomedical Research Ethics Committee of the University of KwaZulu-Natal,and participants provided written informed consent.

PBMCs from a total of 32 HIV-1-uninfected and 28 recently HIV-1-infectedindividuals from the CAPRISA study cohort were available for use in this study.

Sample processing, viral load quantification, and CD4 cell enumeration.PBMCs were isolated by Ficoll-Histopaque (Sigma) density gradient centrifuga-tion from blood within 6 h of phlebotomy and frozen in liquid nitrogen until use.Viral load was determined by using the automated COBAS Amplicor HIV-1Monitor Test v1.5 (Roche). CD4� cells were enumerated by using the Multitestkit (CD4/CD3/CD8/CD45) on a four-parameter FACSCalibur flow cytometer(Becton Dickinson).

Lentivirus production. Lentiviruses containing short hairpin RNAs (shRNAs)expressed under the control of the U6 promoter in a lentiviral vector (pLKO.1)that also confers puromycin resistance were generated in 293T cells as previouslydescribed (22).

Plasmids pLKO.1, pLKO.1/scrambleshRNA (Harvard Institute of Proteomics),and pLKO.1/TRIM22shRNA 3�UTR (5�-CCGGTATTGGTGTTCAAGACTATATCTCGAGATATAGTCTTGAACACCAATATTTTTG-3�; Sigma Aldrich)were used. 293T cells were transfected with a packaging plasmid (pCMVdR8.2dvpr; Addgene), a pRSV-Rev (Addgene) envelope plasmid (VSV-G/pMD2.G;Addgene), and the corresponding pLKO.1 vector.

Viral infection. Peripheral blood mononuclear cells (1 � 106 cells) isolatedfrom healthy HIV-negative donors were placed into a 12-well plate in the pres-ence or absence of CD3.8 antibody (0.5 �g/ml) and incubated for 3 days at 37°Cin 5% CO2. Following stimulation, cells were washed with R10 medium and theninfected with HIV-IIIB (NIH AIDS Reagent Repository) by spinoculation (2 hat 2,500 rpm at 37°C) at 2 � 105 cells/well in a 96-well plate. Virus was subse-quently removed, and cells were washed once and then allowed to incubate foran additional 3 days before analysis of TRIM22 expression by Western blotting.

For Jurkat LTR-G cell experiments (JLTR-G; NIH AIDS Reagent Reposi-tory), cells were transduced by using spinoculation as described above, and thecells were puromycin selected 48 h later. Stably transduced JLTR-G cells in96-well plates (2 � 105 cells/well) were incubated with IFN-�2a (1,000 U/ml;Pestka Biomedical Laboratories, NJ) for 24 h. Cells were then infected byspinoculation with HIV-IIIB. Cells were allowed to incubate for 2 h and thenanalyzed for HIV long terminal repeat (LTR)-dependent green fluorescent pro-tein (GFP) expression by flow cytometry on days 2 and 7. Cell culture superna-tants from day 7 samples were harvested and analyzed by a p24 enzyme-linkedimmunosorbent assay (ELISA) (Becton Dickinson).

Type 1 IFN stimulation of immune cell lines. CEM, Jurkat, and THP1 cells in96-well plates (2 � 105 cells/well) were stimulated with IFN-� for 6 h, whereupongene expression was assessed for TRIM5� and TRIM22.

RNA isolation and analysis. For all samples, RNA was extracted immediatelyafter thawing and counting of PBMCs without in vitro stimulation. RNA wasextracted from 2 � 106 PBMCs by using TRIzol LS reagent (Invitrogen). Thetotal RNA concentration was quantified, and samples were used only if theoptical density at 260 nm (OD260)/OD280 ratio was 1.90 or greater. All RNAsamples were DNase treated. One microgram of total RNA from each samplewas reversed transcribed by using the iScript cDNA synthesis kit (Bio-Rad).

RNA quantitation by real-time PCR. The PCR primers and cycling conditionsused for IFN-�, IFN-�, MxA, huTRIM5�, and TRIM22 real-time quantitativePCR are provided in Table 1. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was determined to be the most suitable reference gene based on PCRefficiency. Each PCR mixture consisted of 3 mmol/�l MgCl2, the respectiveprimers (0.5 pmol/�l for MxA, huTRIM5�, and TRIM22 and 0.25 pmol/�l forIFN-�, IFN-�, and GAPDH), 1 �l Fast Start SYBR green I (Roche), 1 �gcDNA, and water (10-�l total volume). Reactions were run with a Roche Light-Cycler v1.5 instrument (1 cycle at 95°C for 10 min and then 45 cycles of dena-turation, annealing, and extension [Table 1]). To confirm amplification specific-ity, the PCR products were subjected to a melting-curve analysis and agarose gelelectrophoresis. Serial dilutions of cDNA from total RNA were performed foreach target gene. These served as standard curves for quantitative analysis.

Western blotting. Antibodies used in this study were rabbit polyclonal anti-TRIM22 (Prestige, catalog number HPA003575; Sigma), mouse monoclonalanti-Ran (catalog number R4777; Sigma), or rabbit polyclonal anti-IFITM1(catalog number ab70477; Abcam). Antibody-antigen complexes were detectedby using enhanced chemiluminescence reagents (Invitrogen).

Statistical analysis. The generation of dot plots, nonparametric statisticalanalysis, and correlations (Pearson) were performed by using the statisticalprograms Instat Graphpad Prism V.5. and SAS. Values are expressed as medi-ans. Differences between groups were evaluated by using a Student’s t test. Wecorrelated huTRIM5� and TRIM22 to type 1 IFN (MxA, IFN-�, and IFN-�)gene expression values and to each other. Pearson correlations were performedon log-transformed data. Univariate and multivariate generalized estimatingequation (GEE) models were fitted to huTRIM5�, TRIM22, MxA, IFN-�, andIFN-� expressions; viral loads; and CD4 cell counts. Viral loads and expression

VOL. 85, 2011 TRIM22 AND HIV-1 INFECTION 209

levels were log transformed, while square root transformation was applied toCD4 count data to ensure normality.

RESULTS

Relative expression levels of type 1 IFN (IFN-� and IFN-�),MxA, and huTRIM5� in PBMCs from HIV-1-uninfected ver-sus HIV-1-infected subjects. We have previously shown thatHIV-negative patients have higher levels of huTRIM5� thando HIV-1-positive patients (34). However, in matched pre- andpostinfection samples, we did not see a significant dysregula-tion of TRIM5�. This result was surprising, as TRIM5� is atype 1 IFN-responsive gene, and type 1 IFN is dysregulated inprimary HIV-1 infection (15). Furthermore, we found thatwomen at high risk for HIV-1 infection who did not serocon-vert following 2 years of follow-up had significantly higherTRIM5� mRNA levels in PBMCs than did seroconverters(34). A possible explanation for the latter finding is that high-risk nonseroconverter study participants have generally higherlevels of innate antiviral defense mechanisms, perhaps medi-ated through type 1 IFN, thus providing an explanation for ourobservation of elevated huTRIM5� levels among nonserocon-verters. We therefore sought to better understand the relation-ship between TRIM5�, IFN-�, IFN-�, and a type 1 IFN-in-ducible gene, MxA.

We compared mRNA levels of IFN-�, IFN-�, MxA, andTRIM5� in PBMCs from HIV-1-negative versus HIV-1-infectedsamples collected within the first 12 months postinfection.There were 32 individual HIV-1-negative samples availableand 28 HIV-1-infected samples. Samples from HIV-1-positiveindividuals were available at multiple time points postinfec-tion, and samples closest to the 12-month-postinfection timepoint were included in the analyses presented here. Only pa-tients that remained HIV-1 negative upon 2 years of follow-upwere used for this analysis (n � 19). The expression valueswere log transformed to ensure normality. Median expressionlevels between HIV-negative and HIV-positive samples werecompared by using an unpaired Student’s t test. There were nosignificant differences in IFN-� expression between HIV-1-

negative and –positive participants (Fig. 1A). HIV-1-positiveparticipants had significantly higher levels of IFN-� (P �0.0005) and MxA (P � 0.007) (Fig. 1B and D). As we havepreviously reported, HIV-1-negative PBMCs had significantlyhigher levels of TRIM5� than did HIV-1-positive PBMCs (P �0.0001) (Fig. 1C).

We next investigated the relationship between TRIM5� andtype 1 IFN expression in HIV-1-negative and -positive sam-ples. There was no correlation between TRIM5� and IFN-� orMxA (Fig. 1E and G) for both negative and positive timepoints. All HIV-1-negative (n � 32) and HIV-1-positive (n �75) samples available at multiple time points were used for thisanalysis. We found a significant inverse correlation betweenIFN-� and TRIM5� in both HIV-1-negative (r � 0.49; P �0.004) and HIV-positive (r � 0.39; P � 0.0008) samples (Fig.1F). As expected, our data also indicated that MxA is a suitablesurrogate for type 1 IFN induction, because MxA mRNA lev-els showed a significant positive correlation with IFN-� in bothHIV-1-negative (r � 0.8; P � 0.0001) and HIV-1-positive (r �0.81; P � 0.0001) PBMCs. MxA mRNA levels were also sig-nificantly correlated with IFN-� levels in both HIV-1-negative(r � 0.7; P � 0.0001) and HIV-1-positive (r � 0.8; P � 0.0001)samples (Fig. 1H). Thus, in this cohort of individuals at highrisk for HIV-1 infection in a high-prevalence setting, the levelof TRIM5� was higher in HIV-1-negative than in HIV-1-pos-itive PBMCs, and surprisingly, there was an inverse correlationbetween TRIM5� and IFN-�.

Expression of TRIM22 in PBMCs from HIV-1-uninfectedversus -infected subjects and association between type 1 IFNand TRIM22. We next wished to evaluate the expression of theTRIM22 gene, which is located downstream of TRIM5� onchromosome 11 (location 11p15) (32), because it was shownpreviously to be type 1 IFN inducible in vitro and has knownantiviral activity (2). HIV-1-positive participants had highermRNA levels of TRIM22 than did HIV-negative patients (P �0.01) (Fig. 2A). IFN-� mRNA levels positively correlated withTRIM22 expression levels in both HIV-negative (r � 0.91; P �0.0001) and HIV-1-positive (r � 0.9; P � 0.0001) subjects (Fig.

TABLE 1. Primers used in this study

Gene GenBankaccession no. Sequence (5�–3�)a Cycling conditions (denaturation, annealing,

and extension)

MxA NM_0024462 5�-AAGCTGATCCGCCTCCACTT-3� (F) 95°C for 6 s, 60°C for 6 s, and 72°C for 10 s5�-TGCAATGCACCCCTGTATACC-3� (R)

IFN-� NM_000069 5�-GAAACCACTGACTGTATATTGTGTGAAA-3� (F) 95°C for 6 s, 60°C for 6 s, and 72°C for 10 s5�-CAGCGTCACTAAAAACACTGCTTT-3� (R)

IFN-� L41942 5�-AGTCAGAGGGAATTGTTAAGAAGCA-3� (F) 95°C for 6 s, 60°C for 6 s, and 72°C for 10 s5�-TTTGGAATTAACTTGTCAATGATATAGGTG-3� (R)

huTRIM5� NM_033034 5�-AGGAGTTAAATGTAGTGCT-3� (F) 95°C for 6 s, 60°C for 15 s, and 72°C for 6 s5�-ATAGATGAGAAATCCATGGT-3� (R)

TRIM22 NM_006074 5�-GGTTGAGGGGATCGTCAGTA-3� (F) 95°C for 6 s, 60°C for 6 s, and 72°C for 10 s5�-TTGGAAACAGATTTTGGCTTC-3� (R)

GAPDH NM_002046 5�-AAGGTCGGAGTCAACGGATT-3� (F) 95°C for 6 s, 65°C for 6 s, and 72°C for 6 s5�-CTCCTGGAAGATGGTGATGG-3� (R)

a F, forward; R, reverse.

210 SINGH ET AL. J. VIROL.

2B). IFN-� also showed a significant positive correlation withTRIM22 in both HIV-negative (r � 0.93; P � 0.0001) andHIV-1-positive (r � 0.87; P � 0.0001) subjects (Fig. 2C). Like-wise, MxA mRNA levels correlated positively with TRIM22mRNA levels in both HIV-1-negative (r � 0.81; P � 0.0001)and HIV-1-positive (r � 0.92; P � 0.0001) subjects (Fig. 2D).Thus, TRIM22 positively correlates with type 1 IFN expressionin both HIV-1-negative and HIV-1-positive PBMCs in vivo.

Expression of type 1 IFN (IFN-� and IFN-�), MxA, andTRIM22 mRNA in PBMCs at baseline (study enrollment)from nonseroconverters versus seroconverters. We next ad-dressed whether preinfection samples from seroconverters dif-fered from those from nonseroconverters in IFN-�, IFN-�,MxA, and TRIM22 expression levels. Although seroconvertersshowed generally higher mRNA levels of IFN-� and MxA thannonseroconverters, the differences between the groups did not

FIG. 1. Expression of type 1 IFN (IFN-�, IFN-�, and MxA) and huTRIM5� in PBMCs from HIV-1-uninfected versus HIV-1-infected subjectsand association between type 1 IFN and huTRIM5�. The samples from infected participants were all collected within 12 months of infection. Atleast two time points were available postinfection for the primary infection samples. For the HIV-1-positive group we compared one time point,closest to the set point of 12 months postinfection (n � 28). Only patients that remained HIV-1 negative upon follow-up were used for this analysis(n � 19). Data are depicted as the normalized ratio of huTRIM5�, IFN-�, or IFN-� versus GAPDH. The expression values were log transformedto ensure normality. Median expression levels between HIV-negative and HIV-positive samples were compared. The differences between groupswere evaluated by using an unpaired Student’s t test. A P value of �0.05 was considered statistically significant. Pearson correlations wereperformed for huTRIM5� and IFN-�, IFN-�, or MxA for both negative and positive patients. Pearson correlations were also performed for MxAand IFN-� or IFN-� for both negative and positive patients.

FIG. 2. Expression of TRIM22 in PBMCs from HIV-1-uninfected versus HIV-1-infected subjects and association between type 1 IFN andTRIM22. The samples from infected participants were all collected within 12 months of infection (primary infection phase). At least two timepoints were available postinfection for the primary infection samples. For the HIV-1-positive group we compared one time point, closest to theset point of 12 months postinfection (n � 28). Only patients that remained HIV-1 negative upon follow-up were used for this analysis (n � 19).Data are depicted as the normalized ratio of TRIM22 versus GAPDH. The expression values were log transformed to ensure normality. Medianexpression levels between HIV-negative and HIV-positive samples were compared. The differences between groups were evaluated by using anunpaired Student’s t test. A P value of �0.05 was considered statistically significant. Pearson correlations were performed for TRIM22 and IFN-�,IFN-�, or MxA for both negative and positive patients.


reach statistical significance (Fig. 3A and C). Individuals whobecame HIV-1 positive (n � 13) during the 2-year study fol-low-up period had significantly higher IFN-� (P � 0.0001) andTRIM22 (P � 0.0022) mRNA levels preinfection than didthose who remained HIV-1 negative (n � 19) (P � 0.0001; P �0.0022) (Fig. 3B and D).

Association between antiviral gene expression, viral load,and CD4 T-cell counts. To determine if IFN-�, IFN-�, MxA,huTRIM5�, and TRIM22 gene expressions had functional im-plications for viral control during primary infection, we used ageneralized estimating equation (GEE) model to evaluate viralload or CD4� T-cell counts, adjusting for repeated measure-ments for the same individual. In the univariate models, MxAdisplayed a statistically significant association with HIV-1plasma viral load. For every log increase in MxA mRNA levels,the viral load increased by 0.29 log copies/ml (P � 0.0444).IFN-� also showed a positive association with viral load; how-ever, this was not statistically significant (P � 0.0995).

Following adjustment for the other antiviral factors includedin this study, MxA and TRIM22 maintained a statistically sig-nificant association with viral load. The association betweenMxA and HIV-1 viral load increased after adjusting for theother antiviral factor expression variables, with every log in-crease in MxA increasing the viral load by 0.85 log copies/ml(P � 0.0418). On the other hand, for every log increase in

TRIM22, the viral load decreased by 0.98 log copies/ml (P �0.0307) (Table 2).

CD4� T-cell counts are an important correlate of diseaseprogression rate and outcome in HIV-1 infection. We there-fore investigated whether IFN-�, IFN-�, MxA, huTRIM5�,and TRIM22 had any association with CD4� T-cell countsduring primary HIV-1 infection. A GEE model was fitted toCD4� T-cell counts, adjusting for repeated measurements forthe same individual. In the univariate models, MxA, TRIM22,IFN-�, and IFN-� all had significant a negative associationwith CD4� T-cell counts; thus, as the expression increased with1 log, CD4� T-cell counts decreased by 2.12, 1.79, 1.75, and2.04 square root CD4� T cells/�l for these factors, respectively.However, in the multivariate model, only TRIM22 remainedstatistically significant (P � 0.0281), showing a positive associ-ation with CD4� T-cell counts (Table 3).

TRIM22 expression is induced in HIV-negative PBMCs byinfection with HIV-1. We next sought to determine whetherinfection of PBMCs isolated from HIV-negative donors couldinduce TRIM22 expression in vitro. Infection of PBMCs in thepresence or absence of stimulating bispecific CD3.8 antibody(11) resulted in the upregulation of the TRIM22 protein incomparison to uninfected controls (Fig. 4A and B). The stim-

TABLE 2. Association between gene expression and viral loada

GenebUnadjusted model Adjusted model

Effect estimate (SE) P value Effect estimate (SE) P value

MxA 0.2887 (0.1436) 0.0444 0.8539 (0.4195) 0.0418IFN-� 0.2163 (0.1363) 0.1126 0.2512 (0.6476) 0.6981IFN-� 0.2216 (0.1345) 0.0995 0.1693 (0.4987) 0.7343huTRIM5� 0.0368 (0.1308) 0.7785 0.1539 (0.1439) 0.2851TRIM22 0.2154 (0.1532) 0.1597 0.9807 (0.4539) 0.0307

a A GEE model was fitted to viral load, adjusting for repeated measurementsfor the same individual. Unadjusted models were fitted for each expression levelin order to determine the effect on viral load. An adjusted model was fitted,including all expression variables in the model to determine whether they havean effect while adjusting for other expression levels. Viral load and expressionlevel were log transformed to ensure normality. Boldface type indicates signifi-cance.

b Versus GAPDH.

FIG. 3. Expression of type 1 IFN (MxA, IFN-�, and IFN-�) and TRIM22 mRNA in PBMCs at baseline (study enrollment) for nonserocon-verters versus seroconverters. Participants included in this analysis were all enrolled as high-risk HIV-1-uninfected individuals and were longitu-dinally monitored for at least 36 months each at the time of analysis. Data are depicted as the normalized ratio of IFN-�, IFN-�, MxA, andTRIM22 versus GAPDH. The expression values were log transformed to ensure normality. The horizontal line represents the median. Thedifferences between groups were evaluated by using an unpaired Student’s t test. A P value of �0.05 was considered statistically significant.

TABLE 3. Association between gene expression and CD4� T-cellcounts after HIV infectiona

GenebUnadjusted model Adjusted model

Effect estimate (SE) P value Effect estimate (SE) P value

MxA 2.1227 (0.8552) 0.0131 4.1024 (2.3833) 0.0852IFN-� 1.7526 (0.7378) 0.0175 2.9797 (2.4699) 0.2277IFN-� 2.0405 (0.6065) 0.0008 0.8828 (2.3954) 0.7125huTRIM5� 2.0405 (0.6065) 0.0008 0.3175 (0.5683) 0.5764TRIM22 1.7936 (0.8945) 0.0450 6.0974 (2.7761) 0.0281

a A GEE model was fitted to CD4� T-cell counts, adjusting for repeatedmeasurements for the same individual. Unadjusted models were fitted for eachexpression level in order to determine the effect on CD4� T-cell counts. Anadjusted model was fitted, including all expression variables in the model todetermine whether they have an effect while adjusting for other expression levels.A square root transformation was applied to CD4� T-cell counts to ensurenormality. The expression level was log transformed. Boldface type indicatessignificance.

b Versus GAPDH.


ulation of PBMCs with CD3.8 antibody with no infection alsoresulted in a slight increase in the TRIM22 expression level,indicating that activation alone was enough to alter TRIM22expression.

To validate the role of IFN-� in TRIM22 induction, westimulated a number of cell lines used in HIV infection assays(CEM, Jurkat, and THP-1) with increasing amounts of IFN-�and examined TRIM22 and huTRIM5� expression by reversetranscription (RT)-PCR. IFN-� significantly upregulatedTRIM22 and huTRIM5� expression in a dose-dependentmanner in all three cell lines tested (Fig. 4C and D).

Silencing of TRIM22 increases HIV infection and virus re-lease in the presence of IFN-�. To determine its functional rolein HIV infection, we tested the role of TRIM22 in HIV infec-tion. JLTR-G reporter cells were stably transduced with emptypLKO vector, control scrambled shRNA (control), or anshRNA directed against the 3� untranslated region (3�UTR) ofTRIM22 and then challenged with HIV-IIIB (a fully infectiouslaboratory strain), with our without stimulation by IFN-�. Atday 7, the percentage of HIV-infected cells (GFP-positivestaining) in the 3�UTR cells treated with type 1 IFN exhibiteda significantly higher percentage of infected cells (51.2%) thandid vector-treated (22%) and control (24.5%) cells in the pres-ence of IFN-� (Fig. 5A). Furthermore, the percentage of in-fected TRIM22-depleted cells was nearly equivalent regardlessof whether cells had been treated with IFN-� or not, strongly

demonstrating a significant functional role for TRIM22 in theanti-HIV IFN-� response. Data for culture supernatants col-lected on day 7 were consistent with these observationswhen assessed for p24 levels by ELISA (Fig. 5B). Theknockdown of TRIM22 by the 3�UTR shRNA in the pres-ence of IFN-� was validated by both RT-PCR and Westernblotting (Fig. 5C and D).

DISCUSSION

For species other than humans, it has been demonstratedthat restriction factors can completely block or partially restrictretroviral infection (7, 8, 12, 24, 36). In contrast, little is knownabout the in vivo regulation of restriction factors or their pos-sible role in protecting or controlling retroviral infections inhumans. In this study, we used a well-characterized clinicalcohort of high-risk seronegative and acute or primary infectionsamples to investigate the association of the expression ofselect type 1 IFN isoforms, two well-characterized TRIM E3ligases (TRIM5� and TRIM22), and the impact on HIV-1susceptibility and viral control during primary HIV-1 infection.

Our earlier study revealed that lower huTRIM5� mRNAexpression levels were associated with increased susceptibilityto HIV-1 infection in a cohort of high-risk black African fe-males (34). In addition, we found that in matched samples of

FIG. 4. Induction of TRIM22 expression by HIV and IFN-�. Shown is a representative example of data for TRIM22 induction in PBMCs froma healthy HIV-negative donor following infection with HIV-IIIB in the presence or absence of stimulating CD3.8 antibody. (A) Expression ofTRIM22 following HIV infection was assessed by Western blotting. Ran levels are shown as a loading control. (B) Relative fold induction ofTRIM22 by HIV averaged over three HIV-negative donors as determined by densitometric analysis (NIH Image). Values were normalizedto TRIM22 expression following CD3.8 stimulation and HIV infection in each donor. (C and D) Dose-dependent increases in TRIM22 (C) andhuTRIM5� (D) in various immune cell lines as determined by RT-PCR.


HIV-1-negative individuals who later became HIV-1 positive,huTRIM5� levels were not dysregulated following infection.

Here we sought to understand the relationship betweenhuTRIM5� and type 1 IFN in HIV-1-negative and -positivedonor PBMCs, in part because huTRIM5� is type 1 IFN in-ducible (4, 31). Similarly, we also assessed the expression andactivity of the related TRIM22 protein, focusing on this proteinbecause, like TRIM5�, it is IFN inducible and has been dem-onstrated to have anti-HIV-1 activity (2, 4, 25). We found thatPBMCs from HIV-1-positive study subjects had higher levelsof IFN-� and MxA, suggesting that these antiviral proteins areactively upregulated following HIV-1 infection. This result isconsistent with findings from other groups (42, 44). We did notsee significant differences in IFN-� expression between HIV-1-negative and -positive PBMCs, suggesting that there may bedifferences in the mobilization of the varied type 1 IFN iso-forms (10, 23) following HIV-1 infection. However, overall, inboth HIV-1-negative and -positive PBMCs, we found a strongpositive correlation between the two type 1 IFN isoformstested and the IFN-inducible gene MxA, as previously de-scribed (18). Surprisingly, huTRIM5� showed a significant in-verse correlation with IFN-� and had no association withIFN-� or MxA, even though huTRIM5� was shown previously

to be an IFN-�-inducible gene in vitro (31). These results couldreflect the limitations of our methodology here in analyzingglobal expression in PBMCs rather than in specific constituentcellular compartments or using frozen samples instead of freshsamples. Alternatively, we can speculate that distinct type 1IFN isoforms may differentially regulate huTRIM5� expres-sion, or huTRIM5� expression in vivo may involve more-com-plex pathways in addition to type 1 IFN. Further studies areneeded to comprehensively investigate how different type 1IFN isoforms and other cellular proteins may function in theregulation of huTRIM5� in different cellular environmentsand varied cohorts.

Unlike huTRIM5�, TRIM22 mRNA levels were higher inHIV-1-positive participants than in HIV-1-negative ones. Wealso found that TRIM22 correlated with IFN-�, IFN-�, andMxA in both HIV-1-negative and -positive samples. Theseresults show an association between TRIM22 and type 1 IFNexpression in vivo, suggesting that TRIM22 is a type 1 IFN-responsive gene in vivo, as was shown previously in in vitroexperiments (2, 3). Higher levels of IFN-�, IFN-�, MxA, andTRIM22 were detected in seroconverters than in nonserocon-verters at baseline, with differences reaching significance forIFN-� and TRIM22. These results suggest that there is im-

FIG. 5. TRIM22 silencing increases HIV infection and virus accumulation. (A) Jurkat reporter cells (LTR-G) transduced with the empty pLKOvector, control shRNA, or an anti-TRIM22 shRNA targeting the 3�UTR were infected with HIV-IIIB following a 1-day stimulation with IFN-�.Infection of JLTR-G cells (on day 7 postinfection) was assessed by GFP expression using flow cytometry. (B) TRIM22 silencing enhancesaccumulation of HIV particles in culture supernatants as determined by p24 ELISA on day 7 postinfection. (C and D) JLTR-G cells transducedwith the indicated lentivirus were assessed for TRIM22 expression by RT-PCR and Western blotting. IFITM1 was used as a control (3a) for IFNinduction, while Ran was used as a loading control.


mune activation or another dysfunction before these personsbecome HIV-1 positive, and this may have contributed to theincreased susceptibility to infection for these study subjects. Itwas previously demonstrated for a cohort of individuals at highrisk of HIV-1 acquisition that participants who remained se-ronegative had lower levels of CD4� T-cell activation at base-line (when both groups where HIV-1 negative) (16). Similarly,levels of type 1 IFNs are elevated during immune activation,which in turn has been associated with increased HIV-1/AIDSpathology (19, 20). Further analysis of the dynamics of theexpression of these antiviral factors in matched pre- andpostinfection samples, especially from longitudinal cohorts oflow- and high-risk study subjects with frequent sampling, mayfurther help to better understand how intrinsic immunity ismobilized in acute HIV-1 infection and its possible contribu-tion to antiviral control, especially in the critical acute phase ofinfection. It may also be useful to investigate the impact ofHIV-1 infection on the type 1 IFN pathway response genes inmucosal tissues.

We also investigated the association of antiviral gene expres-sion with viral load and CD4 T-cell counts, two commonly usedmarkers of disease progression. We found that MxA mRNAlevels showed a positive association with plasma viral load. Forevery log increase in MxA, the viral load increased by 0.85 logcopies/ml. This is consistent with data from previous studiesthat demonstrated that levels of type 1 IFN increase as the viralload increases (18). Interestingly, we also observed thatTRIM22 has a negative association with plasma viral load anda positive correlation with CD4� T-cell counts. We found thatfor every log increase in TRIM22 mRNA levels, there is anassociated viral load decrease of 0.98 log copies/ml (P �0.0307). TRIM22 also showed a positive association withCD4� T-cell counts, as every log increase in TRIM22 expres-sion was associated with a 6.09 square root increase in CD4�

cells/�l (P � 0.0281).The new findings of a paradoxical elevation of TRIM22

levels in HIV-positive versus HIV-negative PBMCs and thefavorable association of TRIM22 expression with markers ofdisease outcome prompted us to investigate whether TRIM22could be induced by HIV-1 infection of HIV-negative donorPBMCs. Our experiments confirmed that TRIM22 was in-duced by HIV (Fig. 5A and B) and provided an in vitro cor-roboration of results demonstrating that HIV-1-positive sub-jects have higher levels of TRIM22 than do HIV-negativedonors. This was similar to data from the work of Wang et al.(43), who showed that pseudotyped HIV-1 infection couldinduce APOBEC3G expression. Since PBMC populations arecomposed of a number of immune cell types (T cells andmonocytes, etc.), we also demonstrated that IFN-� exerts anenhancing effect on TRIM22 expression in a dose-dependentmanner in CEM and Jurkat T-cell lines and the monocyte cellline THP1 (Fig. 4C).

In addition, we found that the silencing of TRIM22 in aT-cell line nearly completely abrogated the IFN-mediated re-striction of HIV-1 (Fig. 5A). TRIM22 silencing also resulted inan increased accumulation of HIV particles in culture super-natants (Fig. 5B), suggesting a role for TRIM22 in late viralreplication activities, such as viral release or budding. Overall,therefore, our results are in agreement with data from severalstudies that have suggested that TRIM22 is induced by type 1

IFN and that TRIM22 can potently inhibit HIV replicationand release (2, 3, 37).

Together, these data are suggestive of both in vivo and invitro anti-HIV roles for TRIM22, although it is difficult toprove a cause-effect relationship between TRIM22 expressionlevels and viral load or CD4 T-cell count variables. Based onour findings, we speculate that the targeted enhancement ofthe expression of TRIM22 in HIV-1-infected individuals maybe beneficial in reducing the viral load and could be employedas a novel antiviral strategy.

In conclusion, we have demonstrated with a cohort of HIV-1-uninfected and -infected individuals in a high-prevalence set-ting that HIV-1 infection is associated with an increased ex-pression of the antiviral factor genes IFN-� and MxA, keycomponents of the type 1 IFN pathway. However, we did notfind a correlation between IFN-� or MxA and huTRIM5�, apreviously described type 1 IFN-responsive host restrictionfactor. Indeed, we found a significant negative correlation be-tween IFN-� and huTRIM5�. In contrast, we found thatTRIM22 levels strongly correlated with IFN-�, IFN-�, andMxA expression in both HIV-1-negative and -positive PBMCsand were upregulated in HIV-1-positive study subjects. Re-markably, TRIM22 was associated with lower plasma HIVviral loads and higher CD4 T-cell counts in multivariate mod-els adjusted for multiple antiviral factors analyzed, suggestingthat TRIM22 could have antiviral effects in vivo. We show invitro that TRIM22 is induced by type 1 IFN and HIV-1 infec-tion. Furthermore, we demonstrate that TRIM22 plays a crit-ical role in type 1 IFN-induced anti-HIV-1 activity in tissuecultures. This is the first study to provide evidence suggestingan in vivo antiviral activity of TRIM22. Further studies will beneeded to address what specific cell types in the PBMC milieuexpress TRIM22 and the other members of the TRIM family,to better define how the expression of these proteins is regu-lated and to address whether these proteins can be harnessedas antiviral therapies or prophylactics.

ACKNOWLEDGMENTS

The CAPRISA 002 study was supported by the National Institute ofAllergy and Infectious Diseases (NIAID), National Institutes ofHealth (NIH), U.S. Department of Health and Human Services (grantU19 AI 51794). This study was funded by grants from the SwitzerlandSouth African Joint Research Programme (SSAJRP) (J.L. and T.N.),the Hasso Plattner Foundation and the South African DST/NRF Re-search Chair in Systems Biology of HIV/AIDS (T.N.), the NIAID(grant R01AI059159), and grant 3100AO-128655 from the SNF (J.L.).R.S. was a recipient of a KwaZulu-Natal Research Institute for TB andHIV (K-RITH) travel award.

We thank the study participants and the CAPRISA clinical andlaboratory staff for providing specimens. We give special acknowledg-ments to the following members of the CAPRISA Acute InfectionStudy team: Carolyn Williamson, Lynn Morris, Clive Gray, WinstonHide, and Francois van Loggerenberg.

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association of trim22 with the type 1 interferon response

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